Proto-oncogenes and other genes which may be involved in the development or progression of human malignancies are often found to be amplified in many human neoplasms. The overexpression of the gene product in such cases is believed to contribute to tumorigenicity. In vivo, the amplified genes are most frequently detected in acentric, autonomously replicating extrachromosomal molecules. This proposal presents a two pronged approach for the development of methods to eliminate extrachromosomally amplified sequences as a means of reducing tumorigenicity. The first approach extends observations made over the past two years in this Drug Discovery Group that ribonucleotide reductase and topoisomerase inhibitors can accelerate the elimination of amplified proto-oncogenes and drug resistance genes from a variety of cell lines in cell culture to reduce soft agar cloning efficiency and increase drug sensitivity, respectively. Experiments are proposed to test new drugs, alone and in combination, to accelerate the loss rates obtained thus far. An in vivo mouse model will be developed to assess the generality of the in vitro work, and to provide a precedent for implementation of the best strategies in human clinical trials. The molecular mechanisms of elimination will also be investigated to enable the development of additional theoretically based and potentially superior strategies. The second approach will involve the design of molecular reagents which specifically interact with, and inhibit the function of, the replication origin(s) which drive the replication of the extrachromosomal elements. This will be accomplished by the molecular isolation of mammalian replication origins and determination of the minimum sequence necessary for them to function in vivo. Two strategies are proposed to determine whether large regions of DNA are necessary to elicit origin function, or whether small DNA fragments can comprise an origin as long as they are first "imprinted" by integration into a chromosome. The minimum functional regions will be sequenced, analyzed for protein binding sites, and relevant sites mutated in vitro to assess the functional consequences. This information will be used to design and develop origin- specific oligonucleotide inhibitors, and the functional assays will then be used to assess their effectiveness in vitro and in vivo. Together, the two approaches offer a balance between general and highly site specific new strategies for the treatment of human neoplasms in which gene amplification may augment tumor phenotype.